SUPPLEMENTAL DATA AGING, April 2013, Vol.5 No.4

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1 SUPPLEMENTAL DATA Figure S1. Under CR conditions, the atg32δ mutation elevates the extent of oxidative damage to proteins. WT and atg32δ strains were cultured in the nutrient rich YP medium initially containing 0.2% glucose. Immunodetection of carbonyl groups in oxidatively damaged proteins of purified mitochondria was performed as described in Methods. Figure S2. Lipid synthesis in the ER and mitochondria and lipid transport via mitochondria ER and PM ER junctions. After being synthesized in the mitochondria associated membrane (MAM) domain of the ER, cytidine diphosphate diacylglycerol (CDP DAG) and phosphatidylserine (PS) are transported via mitochondria ER junctions to the outer membrane (OMM). Following their translocation from the OMM to the inner membrane (IMM), CDP DAG and PS are used as substrates for the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE), respectively. PE is then transported through mitochondria ER junctions to the MAM domain of the ER, where it is converted to phosphatidylcholine (PC). After being formed in the MAM, PC is translocated via mitochondria ER junctions to mitochondria, which are incapable of synthesizing this glycerophospholipid. The glycerophospholipids phosphatidic acid (PA) and phosphatidylinositol (PI) are also synthesized in the MAM domain of the ER, from which they are delivered through mitochondria ER junctions to mitochondria known to lack enzymes required for the synthesis of these two glycerophospholipids. After being synthesized within the ER and inner membranes, lipids can be translocated via the PM ER junctions from the PM associated membrane (PAM) domain of the ER to the plasma membrane (PM). Unlike glycerophospholipids, various molecular forms of the neutral lipids triacylglycerols (TG) cannot be moved from the site of their synthesis in the ER to either mitochondria or the PM. See text for details AGING, April 2013, Vol.5 No.4

2 Table S1. List of individual protein components of digitonin solubilized membrane protein superes separated by 1 D BN PAGE, resolved by 2 D SDS PAGE and stained by silver. Following silver staining, protein bands were excised from the gel, reduced, alkylated and in gel digested with trypsin. The resulting peptides were separated by RP HPLC/MS using an LTQ Orbitrap. The raw MS data file obtained by Xcalibur was analyzed using the Thermo Scientific Xcalibur Proteome Discoverer application (version 1.3), which was used to identify individual protein components of the isolated respiratory superes by comparing the raw data of mass spectra of digested fragments to the mass spectra of peptides within the Uniprot FASTA database. The analysis by the Thermo Scientific Xcalibur Proteome Discoverer coupled to the FASTA database was enabled by using the peakfinding search engine SEQUEST, which processes MS data using a peak finding algorithm to search the raw data for generating a peak probability list with relative protein abundances. Protein name Wild-type strain Aac2p Por1p Ndi1p Nde1p Pda1p Pdb1p Lat1p Lpd1p Protein description Major ADP/ATP carrier of the inner membrane porin (voltagedependent anion channel) NADH:ubiquinone oxidoreductase external NADH E1 alpha of the pyruvate E1 beta of the pyruvate Dihydrolipoamide acetyltransferase component (E2) of the pyruvate Dihydrolipoamide component (E3) of the pyruvate NCBI accession number Score Sequence coverage (%) Number of identified peptides MW (kda) Protein super 1 P % P % P % P % P % P % P % P % Calculated pi AGING, April 2013, Vol.5 No.4

3 Sdh1p Sdh2p Sdh3p Sdh4p Cyt1p Qcr1p Qcr2p Qcr7p Qcr8p Rip1p Flavoprotein of succinate II) Iron-sulfur of succinate II) Cytochrome b of succinate II) Membrane anchor of succinate II) III), cytochrome c1 component III), 1 III), 2 III), 7 III), 8 III), ubiquinolcytochrome-c reductase Q % P % P % P % P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

4 Atp17p Aac2p Por1p Kgd1p V), alpha V), beta V), gamma V), b V), 5 V), d V), f Major ADP/ATP carrier of the inner membrane porin (voltagedependent anion channel) Component of the alphaketoglutarate P % P % P % P % P % P % Q % Protein super 2 P % P % P % AGING, April 2013, Vol.5 No.4

5 Qcr1p Qcr2p Qcr7p Qcr8p Rip1p Cyt1p Qcr1p Qcr2p Qcr7p III), cytochrome c1 component III), 1 III), 2 III), 7 III), 8 III), ubiquinolcytochrome-c reductase III), cytochrome c1 component III), 1 III), 2 III), 7 P % P % P % P % P % P % Protein super 3 P % P % P % P % AGING, April 2013, Vol.5 No.4

6 Qcr8p Rip1p Atp17p III), 8 III), ubiquinolcytochrome-c reductase V), alpha V), beta V), gamma V), b V), 5 V), d V), f P % P % P % P % P % P % P % P % Q % AGING, April 2013, Vol.5 No.4

7 Aac2p Yme2p Aac2p Major ADP/ATP carrier of the inner membrane V), alpha V), beta V), gamma V), b V), 5 V), d Integral inner membrane protein with a role in maintaining nucleoid structure and number Major ADP/ATP carrier of the inner membrane Protein super 4 P % P % P % P % P % P % P % Protein super 5 P % Protein super 6 P % AGING, April 2013, Vol.5 No.4

8 Cyt1p Qcr1p Qcr2p V), alpha V), beta V), gamma V), b V), 5 V), d III), cytochrome c1 component III), 1 III), 2 P % P % P % P % P % P % Protein super 7 P % P % P % AGING, April 2013, Vol.5 No.4

9 Qcr7p Qcr8p Rip1p Cox4p Cox5Ap Cox5Bp Cox6p III), 7 III), 8 III), ubiquinolcytochrome-c reductase Cytochrome c oxidase IV), 4 Cytochrome c oxidase IV), 5A Cytochrome c oxidase IV), 5B Cytochrome c oxidase IV), 6 V), alpha V), beta V), gamma P % P % P % P % P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

10 Ald4p Por1p Cyt1p Qcr1p Qcr2p Qcr7p Qcr8p V), b V), 5 V), d aldehyde porin (voltagedependent anion channel) III), cytochrome c1 component III), 1 III), 2 III), 7 III), 8 P % P % P % Protein super 8 P % P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

11 Rip1p Idh1p Idh2p III), ubiquinolcytochrome-c reductase V), alpha V), beta V), gamma V), b V), 5 V), d Subunit of NAD(+)- dependent isocitrate Subunit of NAD(+)- dependent isocitrate P % P % P % P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

12 atg32δ strain Aac2p Por1p Lat1p Lpd1p Cyt1p Qcr1p Qcr2p Qcr7p Qcr8p Rip1p Major ADP/ATP carrier of the inner membrane porin (voltagedependent anion channel) Dihydrolipoamide acetyltransferase component (E2) of the pyruvate Dihydrolipoamide component (E3) of the pyruvate III), cytochrome c1 component III), 1 III), 2 III), 7 III), 8 III), ubiquinolcytochrome-c reductase Protein super 1 P % P % P % P % P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

13 Aac2p Por1p Cyt1p Qcr1p V), alpha V), beta V), gamma V), b V), 5 V), d Major ADP/ATP carrier of the inner membrane porin (voltagedependent anion channel) III), cytochrome c1 component III), 1 P % P % P % P % P % P % Protein super 2 P % P % P % P % AGING, April 2013, Vol.5 No.4

14 Qcr2p Qcr7p Qcr8p Rip1p III), 2 III), 7 III), 8 III), ubiquinolcytochrome-c reductase V), alpha V), beta V), gamma V), b V), 5 P % P % P % P % Protein super 3 P % P % P % P % P % AGING, April 2013, Vol.5 No.4

15 Aac2p Aac2p V), d Major ADP/ATP carrier of the inner membrane Major ADP/ATP carrier of the inner membrane V), alpha V), beta V), gamma V), b V), 5 V), d P % Protein super 4 P % Protein super 6 P % Protein super 7 P % P % P % P % P % P % AGING, April 2013, Vol.5 No.4

16 Ald4p Por1p Idh1p Idh2p aldehyde porin (voltagedependent anion channel) Subunit of NAD(+)- dependent isocitrate Subunit of NAD(+)- dependent isocitrate Protein super 8 P % P % P % P % AGING, April 2013, Vol.5 No.4